Project Summary/Abstract (30 lines) MicroRNAs (miRNAs) constitute a large family of short, non-coding, regulatory RNAs. To generate functional miRNAs, primary transcripts (pri-miRNAs) need to be first cleaved by an RNAse III enzyme, Drosha. This critical step of miRNA maturation needs to be strictly controlled, and dysregulation is associated with many diseases. Processing efficiency can also tuned through various means, including post-transcriptional modification of RNA, such as adenosine methylation by Mettl3/Mettl14 complexes. In addition, Drosha is dependent on its partner protein, DGCR8. DGCR8 is regulated at the mRNA and protein levels, and is also regulated by heme binding. However, mechanistic details of how pri-miRNA processing is carried out and regulated remain largely unknown, leaving many questions unanswered: 1) what makes a pri-miRNA a good substrate for processing by Drosha? 2) how are processing rates modulated by changes in RNA modification or conformation? 3) why does Drosha require a cofactor protein (DGCR8), unlike other RNAseIII enzymes? and 4) how do DGCR8 and Drosha cooperate to recognize, bind and precisely cleave substrate RNAs? Our long-term goal is to understand miRNA processing and regulation at the atomic level. The Drosha/DGCR8 complex (also called Microprocessor) must efficiently process thousands of different transcripts, with single-nucleotide precision. Our broad hypothesis is that RNA features?sequence and structure?dictate the processing fate of each individual pri-miRNA. Our proposed study focuses on elucidating a model for how Microprocessor recognizes and interacts with pri-miRNAs, and identifying the features of pri- miRNAs that allow specific pathways to regulate processing. Our specific objectives are to: 1) Determine how N6-methyladenosine (m6A) modification of pri-miRNAs modulates processing efficiency, thereby identifying the pri-miRNA features that are important for Microprocessor activity; 2) determine how DGCR8 in its heme-bound state helps Drosha, thereby dissecting the molecular role of DGCR8 in pri-miRNA processing; and 3) Reveal three-dimensional structures of Microprocessor/pri-miRNA complexes to explain the biochemical observations in the first two Aims. As a class of molecules devoted to gene regulation, miRNAs are involved in every major area of biology, and Microprocessor is required for proper maturation of almost all miRNAs. Our proposed studies will together elucidate how Drosha and DGCR8 cooperate to recognize certain features of pri-miRNAs. In addition to providing a basic physical framework for how Microprocessor works, the molecular mechanisms that we discover will lay the groundwork for investigating various ways to regulate individual miRNAs, opening doors to developing new therapeutic agents to target gene regulation. -1-